Association between trichomoniasis and prostate and bladder diseases: a population-based case–control study

Trichomonas vaginalis infection is one of the most widespread sexually transmitted infections in the world. There are approximately 276 million cases worldwide. Most men remain undiagnosed and untreated because they are asymptomatic. The chronic inflammation induced by persistent infection may increase the risk of developing genitourinary cancers. In this study, we aimed to investigate the association between trichomoniasis and benign prostate hyperplasia (BPH), prostate cancer (PCa), and bladder cancer (BC) in Taiwan. We designed a case–control study by using the database of the National Health Insurance program in Taiwan. We used the International Classification of Diseases, 9th Revision classifications to classify all the medical conditions in the case and control groups. All odds ratios (ORs) and 95% confidence intervals (CIs) were analyzed using multivariable logistic regression to adjust for all comorbidities and variables. From 2000 to 2015, we enrolled a total of 62,544 individuals as the case group and 187,632 as the control group. Trichomoniasis exposure had a significant association with BPH and PCa (adjusted OR: BPH = 2.685, 95% CI = 1.233–4.286, P = 0.013; PCa = 5.801, 95% CI = 1.296–26.035, P = 0.016). The relative risk was much higher if patients had both trichomoniasis and depression (adjusted OR = 7.682, 95% CI = 5.730–9.451, P < 0.001). Men with trichomoniasis had a significantly higher risk of developing BPH and PCa than those without. Healthcare professionals should not only pay more attention to disease treatment, but also to public health education.

Benign prostate hyperplasia (BPH), prostate cancer (PCa), and bladder cancer (BC) are common diseases in the elderly male population. The pathological mechanism of these diseases is not yet fully understood. Inflammation of the prostate, which can cause proliferation of epithelium and stroma, is considered to be related to both BPH and PCa 1,2 . In addition, urinary tract infection (UTI) is significantly associated with genitourinary cancers (GUC), including kidney, prostate, and bladder cancers 3 . Trichomonas vaginalis infection is one of the most common sexually transmitted infections (STIs), accounting for approximately 276.4 million new cases annually 4 . Because most male patients are asymptomatic and remain undiagnosed and untreated, persistent infection may cause chronic inflammation, which may increase the risk of GUC. There is a lack of research into the relationship between T. vaginalis infection and BC; however, some studies have mentioned that T. vaginalis infection may induce proliferation of prostatic epithelial cells and stromal cells 5,6 . Some in vitro studies showed that PCa may be associated with the up-regulation of the expression of genes that can control cell apoptosis or be overexpressed as a proto-oncogene 7,8 . The study from Vienna General Hospital discovered that 29/86 (33.7%) patients with BPH were positive for T. vaginalis on polymerase chain reaction (PCR) testing 9 (Table S1). We defined the date of the first disease diagnosis as the index date. We also used ICD-9-CM codes to identify patients with T. vaginalis infection (Table S1). In contrast, the control groups were patients without BPH, PCa, or BC. Among all patients in the case and control groups, we not only selected patients in a 1:3 case:control ratio, matching based on age and index date, but also excluded (1) women and patients of unknown sex, (2) patient's aged less than 18 years, and (3) those last diagnosed with trichomoniasis within 1 year before the index date (Fig. 1). The matching method was taken propensity score matching, wherein match tolerance was set at 0.15. The propensity score matching was set as using logistic regression in estimation algorithm and nearest neighbor matching in matching algorithm. The options for nearest neighbor were random in matching order, non-replacement, 1 to 3 matching, and no caliper. The comorbidities in our study included hypertension, myocardial infarction, congestive heart failure, cerebral or peripheral vascular disease, dementia, chronic obstructive pulmonary disease (COPD), type 2 diabetes, renal disease, and malignant disease except PCa and BC. We also evaluated depression as one of the comorbidities in our study because it may be associated with some cancers 13 .
Statistical analysis. The statistical analyses were performed using SPSS version 22.0 (IBM Corp, Armonk, NY, USA). A P-value < 0.05 was considered significant. The Chi-squared or Fisher exact test was used to evaluate distributions between the case and control groups. Continuous variables were evaluated using the t test.
Unconditional multiple logistic regression analyses were performed to evaluate the risks of BPH, PCa, and BC associated with trichomoniasis after adjusting for age, insurance premium, comorbidities, season, urbanization, and level of care. Adjusted models with significant covariates were constructed using background selection with the likelihood ratio test.    Fig. 2).

Discussion
We designed this case-control study based on nationwide data from Taiwan NHIRD. We found that T. vaginalis infection was significantly associated with BPH and PCa in a male population. Therefore, T. vaginalis could be a pathogen that induces BPH and PCa. However, there was no significant association between trichomoniasis and BC. Furthermore, patients with both trichomoniasis and depression had 7.682 times higher risk of developing BPH, PCa, or BC. This result suggests that the joint effect of trichomoniasis and depression could increase the risk of BPH, PCa, or BC.  www.nature.com/scientificreports/ The mechanism of T. vaginalis inducing BPH and PCa still remains unclear. Several studies have demonstrated different possible mechanisms. In women, T. vaginalis induces pro-inflammatory cytokine production, including interleukin-6 (IL-6), interleukin-8 (IL-8), and chemokine ligand 2 (CCL2), while attaching to vaginal epithelial cells 14 . A similar inflammatory reaction was also noted in T. vaginalis-infected prostatic epithelial cells in some in vitro studies 5,6 . Repeated cell damage and repair in chronic inflammation is likely to play an important role in inducing BPH 15 . Furthermore, the alteration in cytokine expression during chronic inflammation may have effects on cell growth and proliferation of the prostate epithelium and stroma in BPH 15 . The activated mast cells stimulated by T. vaginalis-infected prostatic epithelial cells can initiate IL-8 and CCL2 expression 5 . IL-8 could be a predictive marker for BPH 16 . Some in vitro studies demonstrated that IL-8 can stimulate fibroblast growth factor 2 (FGF-2), which causes the mitosis of prostate stromal cells 17 . IL-8 could also cause cyclin D1 expression to promote stromal cells proliferation 18 . In addition, CCL2, secreted by the prostatic stroma fibroblast, could promote both BPH and PCa progression 5 .
T. vaginalis possibly induces carcinogenesis of the prostate. The infected prostatic epithelial cells produce IL-6 in chronic inflammation 19 . In early studies, an elevated serum IL-6 level was noted in patients with advanced PCa 20 . The positive correlation between IL-6 receptor expression and cell proliferation has been reported 21 . IL-6 also induces epithelial-mesenchymal transition (EMT) in breast cancer growth and metastasis 22 , and the same reaction may also occur in prostatic epithelial cells 23 . In addition, more than one study has demonstrated that IL-6 could enhance androgen receptor (AR) activity and AR gene expression 24 , which is also related to prostate cancer growth. Twu et al. demonstrated that T. vaginalis macrophage migration inhibitory factor (TvMIF) plays an important role in inducing PCa 7 . There are already studies that have proven that higher human macrophage migration inhibitory factor (HuMIF) levels are present in several cancers, including PCa 25 . The structure of TvMIF is similar to that of HuMIF, which might explain why TvMIF also has the ability to promote cell proliferation, sustain inflammation, and stimulate the growth of prostate cancer cells 7 .
In previous studies, T. vaginalis could play an important role as a carcinogen of female cervical cancer 26,27 . However, there is no consensus regarding the relationship between trichomoniasis and cervical cancer 28 . Likewise, the role of T. vaginalis in the development of PCa is still controversial. Zhu et al. demonstrated that there was a negative association between PCa and trichomoniasis 29 . Instead, they discovered culture supernatant of T. vaginalis not only inhibited growth but also induced apoptosis of prostate cancer cell. T. vaginalis could enhance anti-proliferative molecules and decrease the expression of anti-apoptotic molecule 29 . The T. vaginalis adhesion protein could induce T helper 2 cell cytokines reaction to stimulate the productions of specific antibody 30 . This enhancement of the immune response might suppress the cancer cell activity 31 . Moreover, another further study Table 5. Risk of BPH/prostate cancer or bladder cancer stratified by trichomoniasis and depression status using logistic regression. Adjusted OR adjusted odds ratio (adjusted for variables listed in Table 2), CI confidence interval. www.nature.com/scientificreports/ also showed that T. vaginalis seropositivity does not raise mortality risk in men with PCa 32 . The inflammatory response caused by T. vaginalis might not have influence in the development and progression of PCa 32 . However, the detail mechanism of immune response between T. vaginalis and prostate epithelial cell still remained unclear, further investigations are necessary. There were still a lack of studies to prove that trichomoniasis is associated with BC. We still included BC patients in our study because the inflammatory cytokines found in trichomoniasis, including IL-6 and IL-8, are also associated with a higher risk of developing BC 33,34 and some parasites, such as Schistosoma haematobium, can induce BC. However, our study shows no significant association between T. vaginalis infection and BC probably because of limited sample.
We added depression as one of the comorbidities in our study due to another previous nationwide populationbased cohort study in Taiwan which showed that patients with trichomoniasis had higher risks for developing an individual psychiatric disorder, including depression, anxiety, bipolar disorder, schizophrenia and substance abuse 35 . Our study results demonstrate that except for depression, no comorbidities had a significant association with BPH, PCa, or BC. The joint effect of trichomoniasis and depression increased the risk by 7.682 times that of the control group. A recent study showed that depression is associated with decreased immunity 36 . Moreover, depression can also cause cytokine dysregulation and increased serum IL-6 concentration 36 , which might enhance carcinogenesis after T. vaginalis infection.
Although this study was a large-scale population-based nationwide design with long-term monitoring from 2000 to 2015, there are still several limitations. First, the NHIRD does not contain detailed information regarding the symptom severity of BPH, the histological and TNM classification of PCa and BC, serum sex hormone concentrations, Prostate-Specific Antigen (PSA) levels, T. vaginalis antibody test, family history, or personal history such as sexual exposure, physical activity, alcohol consumption or tobacco smoking. Second, we did not include body mass index (BMI) as one of our variables. Obesity is one of the risk factors for BPH and PCa 37 , which might affect their association with trichomoniasis. Third, our study might underestimate the exact number of patients with trichomoniasis. Most male patients would not seek treatment due to being asymptomatic, and ineffective screening protocols because of the lack of public health awareness could also lead to possible T. vaginalis infection being neglected 38 . Another reason that caused underestimation of our case group is that the antibody tests of T. vaginalis were not performed popularly during diagnosis and mostly were female patients 39 . It is possible that T. vaginalis was substantially undercoded and underrepresented in the study population. Fourth, the number of cases of BC might be too small to be significant and the tracking time might not be sufficient for disease monitoring. Trichomoniasis can be a chronic infection. The outcomes in the study might present later in life, so in some men trichomonas exposure may happen a few years before these outcomes appear or many decades prior to diagnosis. Fifth, the outcome of each case was defined as the first code for BPH, PCa, or BC. This assumes that there is a common pathway between trichomoniasis and these 3 separate diseases. However, this approach method could also ignore these outcomes as comorbidities. For example, patients with PCa or BC could also have BPH or other urinary symptoms. It is possible that many PCa or BC outcomes were ignored if BPH was coded first. This might be another reason that our study samples were underestimated. Sixth, our study was designed as an observational case-control study, so the causation cannot be detected. We hope that in the future more research will support our thesis.

Conclusion
Male patients with T. vaginalis infection have an increased risk of developing BPH and PCa, especially in trichomoniasis patients with comorbid depression. Due to the lack of awareness of this pathogen, clinicians should not only treat patients who are already diagnosed but should also pay more attention to groups with higher trichomoniasis exposure risk.

Data availability
Data supporting the conclusions of this article are included within the article and its additional files. The datasets used and/or analyzed during the present study will be made available by the corresponding author upon reasonable request.